Physcomitrium patens and have a major role in the development of this species. Δarfddub protonemata exhibit a delay in filament branching, as well as a delay in the chloronema to caulonema transition. Additionally, leafy gametophore development in Δarfddub lines lags behind wild type. We present evidence that ARFd1 interacts with activating ARFs via their PB1 domains, but not with repressing ARFs. Based on these results, we propose a model in which clade-D ARFs enhance gene expression by interacting with DNA bound clade-A ARFs. Further, we show that ARFd1 must form oligomers for full activity.Auxin response factors (ARFs) are a family of transcription factors that are responsible for regulating gene expression in response to changes in auxin level. The analysis of ARF sequence and activity indicates that there are 2 major groups: activators and repressors. One clade of ARFs, clade-D, is sister to clade-A activating ARFs, but are unique in that they lack a DNA-binding domain. Clade-D ARFs are present in lycophytes and bryophytes but absent in other plant lineages. The transcriptional activity of clade-D ARFs, as well as how they regulate gene expression, is not well understood. Here, we report that clade-D ARFs are transcriptional activators in the model bryophyte  Auxin Response Factors (ARFs) are plant transcription factors that regulate gene expression in response to changes in auxin level; this study shows that D-clade ARFs, which lack a DNA binding domain and are found in lycophytes and bryophytes, are required for auxin-regulated transcription in the model moss Physcomitrium. The phytohormone auxin is a key regulator of many developmental processes in plants. Auxin is perceived within the nucleus by a co-receptor complex consisting of a TRANSPORT INHIBITOR RESISTANT 1/AUXIN F-BOX (TIR1/AFB) protein, the substrate binding subunit of an SCF E3 ubiquitin ligase complex, and an AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) protein, a transcriptional repressor. Auxin promotes the TIR1/AFB and Aux/IAA interaction resulting in Aux/IAA ubiquitination and degradation ,3.ARFs are classed as transcriptional activators or repressors based on experimental evidence as well as sequence homology ,4. Most Physcomitrium patens [P. patens spores germinate to form filaments comprised of cells called protonemata. Protonemal filaments lengthen via divisions of a tip-growing apical cell. Early in development, the apical cell is slow growing and chloroplast-rich, generating short cells with perpendicular cross-walls as it divides. These cells are referred to as chloronemata. Within days, the apical cell transitions to a fast-growing, chloroplast-poor caulonemal cell. These cells are longer than chloronemal cells, and their cross-walls are at an oblique angle to the axis of growth. The chloronema–caulonema transition is promoted by the auxin indole acetic acid (IAA) based on work in suspension cell culture with another moss, Funaria hygrometrica [Several recent studies have focused on the conserved elements of auxin signaling in bryophytes –9. Much m patens ,11. Auxim patens , phyllidm patens , and colm patens . Haploidometrica . Asymmetometrica ,17.P. patens development. RNAi knock-downs of the PpAFB genes results in plants with striking growth defects including the lack of recognizable caulonemata [P. patens plants lacking Aux/IAA transcriptional repressors, and thus constitutively responding to auxin, make protonemata that could not be clearly identified as either chloronemata or caulonemata [Studies of the auxin co-receptor AFB proteins as well as the transcriptional repressor Aux/IAAs provide further evidence for auxin’s role in lonemata . Similarlonemata . Intereslonemata . This suMarchantia polymorpha has a single activating ARF (MpARF1), a single repressing ARF (MpARF2), and one clade-C ARF (MpARF3) that acts as an auxin-independent transcription factor [M. polymorpha encodes a noncanonical ARF called MpncARF that lacks a DBD [The role of individual ARF proteins in bryophyte growth and development is an active area of study. The liverwort n factor ,19. MpARks a DBD . Despiteks a DBD . HoweverM. polymorpha, the P. patens genome encodes 7 clade-A, 4 clade-B, 3 clade-C, and 2 clade-D ARFs [P. patens clade-D ARFs are homologous to MpncARF and also lack DBDs. Since clade D nomenclature is consistent with the widely accepted clade-A, -B, and -C designation, we propose the use of clade-D (ARFd) to describe this group of ARF proteins [P. patens development is unknown.In contrast to e-D ARFs ,21. The proteins –20. Phylproteins –20, suggHere, we show that the PpARFds play a significant role in auxin response and development. Further, we present evidence that multimerization of the ARFds is key to their function. Lastly, we show that auxin signaling in protonemata plays a role in filament branching, and that the PpARFd’s are required for this process.P. patens auxin-signaling pathway, we screened for mutants resistant to the synthetic auxin, 1-naphthaleneacetic acid (NAA). Seven-day-old tissue regenerated from a homogenized Gransden ecotype strain of P. patens containing the DR5:DsRed2 auxin-responsive reporter as well as 35S:NLS-GFP-GUS [DsRed2 Gd/NLS4) [nar) phenotypes. Wild-type plants produce rhizoid-like cells on this medium, whereas nar mutants produce green protonemal tissue or leafy gametophores , IAA1a (6), IAA1b (4), and IAA2 (5) [DR5 element was tandemly repeated approximately 60 times. This is a possible explanation for the instability of the reporter.To identify novel components of the -GFP-GUS  gene (DRGd/NLS4)  was mutatophores . AlthougIAA2 (5) ,23. TwelnarD122, narD157, and narC18, with lesions in the Pp3c9_21330 locus. This locus encodes a clade-D ARF designated as PpARFd1  and in the narD122 line using CRISPR/Cas9 . To determine the contribution of ARFd2 to the auxin response, we used CRISPR/Cas9 to delete ARFd2 in the DR5 background. For this experiment, the DR5:DsRed2 Gd/NLS4 [DR5-carrying progeny was backcrossed to Reute 2 additional times. For the remainder of the manuscript, this DR5 line is the only one used. After recovering the Δarfd2-8 allele, we used CRISPR/Cas9 to delete ARFd1 from both Δarfd2 and the same DR5 background. In this way, we generated single Δarfd1-18 and Δarfd2-8 mutants as well as the double knockout  reduces auxin levels, resulting in small plants with few gametophores [P. patens mutants with reduced auxin signaling via up-regulation of a clade-B repressing ARF [arfd lines, we counted gametophores in the presence or absence of NAA  and a line lacking all 4 AUXIN F-BOX genes   . We trea1,2,3,4) . Neitherarfd1T653L mutation identified in the narD122 line  background. We refer to this line as arfd1T653L-mYPet. Similarly, we generated lines containing a Y643* mutation (arfd1Y643*-mYPet), which generates a premature stop codon just before the PB1 domain  and ARFd1-mYPet lines had 37% as much chlorophyll on mock medium compared to NAA-supplemented medium. Similar to the results presented in Δarfddub lines were only slightly affected by NAA treatment. Likewise, independent arfd1Y643*-mYPet lines displayed a similar level of NAA resistance indicating that the PB1 domain is necessary for ARFd1 function.Our results indicate that the clade-D ARFs are positive regulators of auxin signaling despite the fact that they lack a DBD and presumably cannot bind DNA directly. To gain insight into how the ARFds regulate transcription, we utilized the 122 line . To conf1 domain . Interesd1-mYPet . We deted1-mYPet . Tissue arfd1T653L-mYPet lines exhibit an auxin sensitivity that is intermediate between the control and Δarfddub lines. Therefore, we conclude that the arfd1T653L protein retains some activity. The arfd1T653L lines exhibit intermediate gametophore development phenotypes as well. They develop gametophores at levels comparable to control plants . These observations suggest that the ARFd proteins are required for the development of faster-growing caulonemata. However, on ammonium-deficient medium (BCD), we do not detect a difference in colony circularity  and only slight differences in distribution  compared to the parental DR5 line  and the percentage of cells with cross walls less than 110° . Conducting a Kolmogorov–Smirnov test on the distribution of cross-wall data determined that the detected shift was significant.In contrast, the presence of ammonium in the medium had a significant effect on caulonemal development. In these conditions, the average cross-wall angle for loronema . MeanwhiRSL6, which is strongly induced by IAA treatment, has a 4-fold higher expression level in tissue grown on BCD   and first subapical (39.6%) cell  . Less of (68.4%) . Exposur6%) cell . This obΔarfd1-18 and Δarfd2-8 had only slight and statistically insignificant changes in branch timing compared to control  compared to 17.3% in the DR5 line. Additionally, 7% to 8% of mutant filaments had branches emerging on or after the fifth subapical cell, compared to only 1.3% of filaments in the DR5 line. Similar to DR5 plants, we observed a stimulation of branch emergence in Δarfddub lines grown on 5 μM IAA  assays in P. patens protoplasts. Interestingly, we observed interaction as subnuclear puncta  that facilitate oligomerization through surface charge interactions in a head to tail fashion ,6. For er puncta .T653L interacted with PpIAA1a as well as full-length ARFd1 in both yeast and in split YFP experiments, arfd1T653LΔDIV did not  is sufficient to enable interaction with ARFa8 and IAA1a and partial ARFd1 function in plants. The fact that both faces are required for full activity strongly suggests that ARFd oligomerization contributes to its activity.P. PATENS INTER-GENIC1 (PIG1) locus [Our interaction and genetic data strongly suggests ARFd1 functions via oligomerization of clade-A ARFs via the PB1 domain. However, these data do not rule out the possibility that clade-D ARFs function to titrate the Aux/IAA repressors away from auxin-responsive promoters . Indeed,1) locus . We recopUbi:mYPet-ARFd1 lines were hypersensitive in terms of chlorophyll content and gametophore production when challenged with 5 μM NAA for 21 days, supporting the hypothesis that ARFd1 activates transcription . Notably, this region includes the B3 repression domain (BRD) motif KLFG. BRD-like motifs facilitate the interaction between ARFs and the co-repressor TOPLESS [pUbi:mYPet-ARFd1b2 lines displayed a similar reduction in chlorophyll level as Δarfddub#3. Likewise, the number of gametophores that developed on pUbi:mYPet-ARFd1b2 colonies did not change in response to NAA. These results indicate that MR of ARFd1 is required for its function. Finally, to determine if the PB1 domain of ARFd1 is sufficient to restore auxin signaling to Δarfddub plants, we expressed the C-terminal 265AA of ARFd1 (PB1d1). This region is downstream of the intrinsically disordered MR and contains the PB1 domain. If the primary function of clade-D ARFs is to titrate the Aux/IAAs away from the promoter region, we would expect pUbi:mYPet-PB1d1 to phenocopy pUbi:mYPet-ARFd1. Instead, the effect of NAA on chlorophyll level in these lines was similar to the pUbi:mYPet-ARFd1b2 lines. While pUbi:mYPet-PB1d1 lines produce more gametophores than the other transgenic lines, they did not develop as many as wild type. Taken together, our data strongly suggest that clade-D ARFs function by activation after oligomerization with clade-A ARFs rather than by titration of Aux/IAA repressors.As a control, we tested full-length mYPet-ARFd1 also under control of the maize ubiquitin promoter . The pUbcription . Interesanalysis , this su TOPLESS . When trM. polymorpha, found that loss of the single clade-D ARF results in effects on auxin-regulated transcription [P. patens.Unlike other members of the ARF family of transcription factors, the clade-D ARFs lack a DBD. Previous work with another bryophyte, cription . HoweverP. patens development because Δarfddub colonies have a higher circularity than wild type. Previous work has associated high colony circularity with decreased auxin signaling. An increase in colony circularity has been interpreted as the reduced ability to make caulonemata [Δarfddub plants can make wild-type levels of caulonemata on ammonium-deficient BCD medium. However, Δarfddub plants grown on ammonium-supplemented BCDAT medium exhibit a delay in caulonemal development. This suggests that a nutrient-sensing developmental pathway masks the developmental phenotypes of Δarfddub in some conditions. Interestingly, even strong auxin-signaling mutants show a nutrient-dependent chloronema–caulonema transition. Future work will determine if the nutrient-dependent pathway interacts with the auxin-signaling pathway, or if it is a parallel genetic pathway in the chloronemal-caulonemal transition.Clade-D ARFs have a clear role in lonemata ,14,18. WΔarfddub plants. We observed a similar branching delay in strong auxin signaling mutants . These data suggest that protonemal branch formation is an auxin-regulated process. These findings corroborate earlier studies in Funaria in which they found an increase in caulonemal branches in response to auxin treatment [pina,pinb mutants and NAA treatment in Physcomitrium found no connection between auxin and caulonemal branches [Independent of ammonium levels, protonemal branching is delayed in reatment . In suppbranches . In our branches . Taken tΔarfddub plants exhibit delayed gametophore development. Interestingly, apart from this delay, Δarfddub gametophores are phenotypically wild type. This suggests that the role of clade-D ARFs in gametophore development is restricted to initiation. In line with these observations, we detected an increase in ARFd1-mYPet levels in the protonemal cell at the base of young gametophore buds. While Δarfddub plants have an ammonium-dependent delay in the chloronemal-caulonemal transition, we did not detect a significant difference in ARFd1-mYPet level between the 2 cell types regardless of ammonium levels in the medium. Additionally, ARFd1 transcript levels are unaffected by the ammonium levels in the 2 media types. In general, the Δarfddub phenotypes are similar to those of the strong iaa2-mDII and afb1,2,3,4 mutants, but slightly weaker. This leads us to propose that clade-D ARFs are involved in general auxin-mediated gene activation events  and enhance transcriptional activation. Indeed, ectopic expression of chimeric ARFd1 with the BRD-containing MR of PpARFb2 does not restore auxin response to the Δarfddub mutant. This indicates that the MR of clade-D ARFs is important for gene activation. Further, ectopic expression of the ARFd1 PB1 domain alone does not restore auxin response to Δarfddub plants. Arfd1T653L/Δarfd2 plants have intermediate phenotypes between ARFd1/Δarfd2 and Δarfddub, suggesting that ARFd1-mediated transcriptional enhancement can occur through dimerization. However, for wild-type levels of auxin signaling ARFd1 has to multimerize with additional factors. One possible scenario is that ARFd1 recruits additional activating ARFs, either ARFa or ARFd, through their PB1 domains  was sterilely homogenized in water in a Waring blender with an autoclaved MC1 mini container, spread onto BCDAT plates containing 100 mg/l gentamicin and 25 mg/l G418 overlain with 90 mm cellophane sheets (AA Packaging) and grown for 1 week. In the course of this work, it was discovered that the gentamicin resistance cassette, 35S:aacC1, confers resistance to both gentamicin and G418. Plates were then placed in a Stratalinker UV Crosslinker (Stratagene) and, with lids removed, irradiated with doses of 254-nm light. The plates were closed, taped, and stored in the dark for 24 h to block photoreactivation of pyrimidine dimers. For the UV dose-response evaluation, 10 doses ranging from 0 to 500 mJ/cm2 were tested before a dose of 150 mJ/cm2 was chosen for mutagenesis because it caused a mortality rate of approximately 40%. After UV and dark treatments, the cellophanes were transferred to BCD plates with 15 μM NAA (dose-response treatments), 1 μM NAA (“narB” round), or 5 μM NAA (“narC” and “narD” rounds), and 5 μM NAA proved to be the most effective concentration and all mutants were isolated from 12 (narC) and 18 (narD) plates. After 2 to 4 weeks, the plates were screened for mutants that produced green protonemata and/or leafy gametophores. A few cells of 224 candidate mutants were spotted onto new BCD plates containing 5 μM NAA and reevaluated after 2 weeks. This yielded 57 mutants . The initial 57 mutants were screened for degron mutations in all 3 AUX/IAA genes  and loss-of-function mutations in DIAGEOTROPICA (DGT/ Pp3c1_32280) by sequencing PCR products. Eighteen new alleles of these loci were identified: narC32, dgt-splice (g to a 7 bp before ATG); narD138, dgt-S117L; narD154, dgt-S106C; narC15, iaa1a-P323L; narD69, iaa1a-P323L; narD75, iaa1a-P323L; narD148, iaa1a-G320S (weak); narD151, iaa1a-P323L; narD153, iaa1a-P323L; narC33, iaa1b-P342F; narC37, iaa1b-P341L; narD68, iaa1b-P342L; narD152, iaa1b-P341L; narC8, iaa2-P328L; narC10, iaa2-P328L; narC36, iaa2-P328F; narD139, iaa2-P328L; narD187, iaa2-P327S. The remaining mutants were compared after growth on BCDAT, BCD, BCD + 0.5 μM NAA, and BCD + 12.5 μM NAA, and 12 were selected based on growth and DR5 expression phenotypes.The Quick-DNA Plant/Seed Miniprep Kit (Zymo), and 7.1 to 8.5 total gigabases of paired-end 100 bp reads (BGI Americas) were generated for each mutant plus the DR5:DsRed/NLS4 parent strain. Using Bowtie 2 with “end-to-end” and “very-sensitive” settings , the res . The vas  using Ss .pGILDA and pB42AD yeast-2-hybrid vectors (Clonetech) via LR Gateway Reactions (Thermo Fisher). To introduce T653L and Y643* mutations into ARFd1 entry clone, standard oligomer-driven mutagenesis protocols were used. See arfddub complementation, ARFd1 truncations and domain swaps were cloned with GoldenGate cloning. Both ARFd1 (full length) and PB1d1  sequences were amplified from pENTR-ARFd1 introducing BsaI and unique 4-nucleotide overhangs via PCR. For ARFd1b2, we generated 3 GoldenGate-ready fragments via PCR off coding sequences cloned into pENTR- 1–778 bp of ARFd1, 1,214 to 1,910 bp of ARFb2, and 1,470 to 2,250 bp of ARFd1. All coding sequence fragments were arranged via GoldenGate with pre-cloned N-terminal mYPet and ZmUbi promoter. The destination plasmid contained a Hsp terminator, 35S:nptII resistance cassette, and homology arms to target the construct to the PIG1 locus. Constructs were transformed into the arfddub#3 line with a CRISPR/Cas9 plasmid targeting the PIG1 locus. Trasformants were selected for with Kanamycin sulfate, and stable lines were screened for expression via mYPet signal.For ARF cloning, cDNA was synthesized from RNA extraction via RNeasy plant mini kit (Qiagen) and SuperScript III reverse transcriptase (Thermo Fisher). Coding sequences were amplified and cloned into pENTR/D-TOPO (Thermo Fisher), and subsequently cloned into pMiniT using the PCR Cloning Kit (New England BioLabs), following the manufacturer’s instructions. The resulting plasmids (pMnVN-Gate and pMnVC-Gate) made N-terminal fusions of either the N or C terminal half of mVenus to our protein-of-interest without the excess sequences needed for Agrobacterium transformation. Given that each plasmid is Gateway (Thermo Fisher) compatible, we were able to use LR reactions with the same entry clones used in Y2H described above. We then transformed 15 μg of each plasmid into P. patens protoplasts. These protoplasts were set to incubate in the light for approximately 60 h before imaging via confocal microscopy.To generate split YFP plasmids, we amplified the N-terminal and C-terminal split YFP cassette described in . We clonMethanol chlorophyll extractions were performed at room temperature overnight in 2 mL Eppendorf tubes on whole tissue pressed lightly between paper towels to remove excess water. Absorbances were measured on a SmartSpec Plus spectrophotometer. Chlorophyll content was calculated via the following equation, as described previously :μgChl/T tests were performed in Excel (Microsoft), and the Kolmogorov–Smirnov, ANOVA, and TukeyHSD test was done with R. Confocal microscopy was performed on a Zeiss LSM 880, with image processing and quantification performed in ImageJ.Images of spot inoculations were taken with a Nikon SMZ1500 microscope and a DS-Ri1 camera. For the colony morphology experiments, pictures were acquired via flatbed scanner. Shape parameters were measured in ImageJ based on a binary mask made from color pictures of plants. For regenerated protoplast assays, images were acquired with BZ-X810 all-in-one microscope (Keyence) using CFP and TRITC filter cubes. Whole plant area was measured in ImageJ using a mask generated from the signal from the calcofluor cell wall stain . Cell length, cross-wall angle, and branch positioning were manually measured in ImageJ. Student’s Dot plots were generated with an internet application . Bar ploP. patens ADENINE PHOSPHORIBOSYLTRANSFERASE (APT) gene (Pp3c8_16590V3.1) was used as a reference housekeeping gene as described [cDNA generated from 3 biological replicates for each condition, with 2 technical replicates each, were run on a CFX Connect Real Time System thermocycler/plate reader (BioRad). A escribed .S1 FigPpARFd1 and PpARFd2, including the targets of CRISPR/Cas9-mediated knock out. (B) Schematic of PpIAA2 and PpAFB mutations. (A) A single nucleotide mutation (red text) in the degron (DII) motif in PpIAA2 results in a stabilized auxin repressor. (C) Alleles recovered when generating afb1,2,3,4 line. Green text indicates endogenous start codon. Start codon was knocked out in PpAFB3. The 2 nt deletion in PpAFB4 generates a frameshift mutation, resulting in a premature stop codon. The afb4 peptide is 21 amino acids long.(A) Models of (PDF)Click here for additional data file.S2 Fign ≥ 18 across 3 replicates. Error bars are standard deviation. (D) Morphology of 21-day-old colonies of arfd mutants are similar on low-nitrogen BCD media. On BCDAT, however, the arfddub are significantly more circular. Letters indicate statistical groups as determined by a TukeyHSD post hoc test of an ANOVA. (E) RSL6, an auxin-induced gene, is significantly up-regulated in tissue grown on BCD for 3 days. Expression level normalized to gene expression on tissue kept on BCDAT for 3 days. IAA2 is nominally down-regulated in repsonce to BCD treatment, while ARFd1 is not differentially expressed. Bar graphs indicate mean of 3 biological replicates, error bars are standard error of the mean. ** = p < 0.005. The underlying data for panels B, C, D, and E are in (A) Images of 21-day-old spot inocula on BCD medium. (B) Normalized chlorophyll content of 21-day-old colonies grown on BCD supplemented with the indicated concentration of NAA. (C) Gametophore counts of 21-day-old colonies grown on BCD supplemented with the indicated concentration of NAA. For both B and C, (PDF)Click here for additional data file.S3 FigpARFd1:PpARFd1-mYPet locus with the PB1 domain in blue. Indicated location of sgRNAs (red text) used to create T653L and Y643* mutations. Scale bar is 1,000 nts. (B) Sequences of WT, narD122, mutating oligomer, and recovered arfd1T653L or arfd1Y643* mutants. Magenta text highlights codon change, with green highlighting silent mutations intended to introduce restriction enzyme sites. Red/pale red text indicates the protospacer-adjacent motif (PAM), while orange text highlights silent mutations introduced to abolish sgRNA binding. For arfd1T653L, only the PAM sequence needed to be changed. For arfd1Y643*, there were no available silent mutations for the lysine residue that would not also disrupt the intron-exon slice site. (C) Micrographs of ARFd1-mYPet and 2 independent lines of arfd1T653L-mYPet. Magenta is chloroplast autoflouresence, yellow is mYPet signal. Often, both ARFd1 and arfd1T653L formed single subnuclear focus (white arrow head) and scale bar is 5 μm. (B) mYPet signal, normalized to ARFd1-mYPet. Signal of arfd1T653L-mYPet (n ≥ 66 cells across 2 replicates) is roughly double that of ARFd1 (n = 170 cells across 2 replicates). * = p ≤ 0.001. The underlying data for panel D are in (A) Gene diagram of (PDF)Click here for additional data file.S4 Figiaa2-mDII, and afb1,2,3,4 each exhibit changes in morphology in response to ammonium in the medium. Scale bar is 500 μm. (B) Cross-wall angles of protonemata from WT (n = 660), iaa2-mDII (n = 631), and afb1,2,3,4 (n = 587) plants across 3 biological replicates grown on BCD medium. Protonemata from both mutant lines have a smaller average cross-wall angle compared to wild type . (C) Protonemata form both lines are enriched in chloronemata . (D) Cross-wall angles of protonemata from WT (n = 583), iaa2-mDII (n = 583), and afb1,2,3,4 (n = 560) plants across 3 biological replicates grown on BCDAT medium . (E) Protonemata form both lines are enriched in chloronemata . The underlying data for panels B, C, D, and E are in (A) Representative micrographs of 7-day-old plants regenerated from single protoplasts. WT, (PDF)Click here for additional data file.S5 Figarfd mutants grown on (+) or off (-) 5 μM IAA for 3 days. (B) Representative micrographs of protonemal filaments of 7-day-old regenerated protoplasts. White arrowhead indicates filament branch. Scale bar 50 μm. (C) Protonemal branching occurrence by cell position. Plants were scored after growing for 3 days on BCD, having regenerated from single protoplasts on PRMB for 4 days. There is a significant delay in the stabilized iaa2-mDII (n = 228 cells) line as well as a line lacking any F-box auxin co-receptor, Δafb1,2,3,4 (n = 144 cells) compared to wild type (n = 329 cells) . (D) Complete table of values of data presented in (C). The underlying data for panels C and D are in (A) Complete table of values for (PDF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 DataT653L-mYPet signal quantification. White outline is ROI for nucleus. Red outline is ROI for background subtraction. Only 1 background ROI was measured for each micrograph containing multiple nuclei. Scale bar is 5 μm.(A) Representive cross-wall measurements. Scale bar is 5 μm. (B) Representive cross-wall measurements and Integrated Density (ID) quantification of ARFd1-mYPet signal in the nucleus (white outline). Scale bar is 5 μm. (C) Representitive example of imaging processing for colony morphology quantificiation. (D) Representitive micrographs of nuclear ARFd1-mYPet and ARFd1(TIFF)Click here for additional data file. 17 Nov 2022Dear Mark, Thank you for submitting your manuscript entitled "Clade-D Auxin Response Factors are Major Regulators of Auxin Signaling in Physcomitrium patens." for consideration as a Research Article by PLOS Biology.Your manuscript has now been evaluated by the PLOS Biology editorial staff as well as by an academic editor with relevant expertise and I am writing to let you know that we would like to send your submission out for external peer review.However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. 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For instructions see: Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Ines--Ines Alvarez-Garcia, PhDSenior EditorPLOS Biologyialvarez-garcia@plos.org------------------------------------------------------------------Reviewers’ commentsRev. 1:The manuscript by Bascom Jr. et al. presents a functional analysis of the D-class ARF proteins in Physcomitrium patens. It combines a genetic approach following a genetic screen for NAA-resistant (NAR) phenotypes, with phenotyping, expression and protein interaction analysis. It is neat and straightforward.The authors identified in the screen three alleles in ARFd1 and constructed the other alleles by CRISPR, and the double mutant d1 d2 also by CRISPR.Here I was confused with the labeling of the genotype in Fig S1 and Figs 2, 3, 4 (and others). In panel A of S1, are presented the gene deletion in arfd2-8, d2-8 d1-1 and d2-8 d1-3. But in panel B, the images are from d1 d2-1 and d1 d2-3. Can the authors checking the accuracy of the labeling in Fig S1 and 2? Maybe a table can be proposed to detail the genotypes used in the study?The authors analysed the effects of the mutations in the PB1 domain, the effects of NAA applications and high-nitrogen. They showed that there is an impact of the mutations of the timing of development of the branches.To further understand how D1 can act on activate transcription without having a DNA binding domain, the authors investigated its interaction with A-ARF and IAA1, by yeast two-hybrid and BCIF, proposing a model that D-ARF act as an enhancer of the activity of A-ARF on the promoters of auxin-responsive genes.The authors indicated that D1 also interacts with A4, while only A8 is shown in Fig 7. I am also curious if the authors looked into the expression pattern (tissue and cell level) of the arfd1 T653L - mYpet. Is the mYPet protein expressed? Is there any differences with the WT D1-mYPet?These are only changes amendments. The manuscript is in state to be published when this is clarified.Rev. 2:In this manuscript, Bascom and colleagues address the function of Clade-D ARFs, a class of ARFs missing an obvious DNA-binding domain which is found only in bryophytes and lycophytes among extant land plants. To this end, they subject the Physcomitrium patens genes ARFd1 and ARFd2 to a functional characterization.The manuscript is well written and to-the-point. Despite a relatively limited set of experiments and a compact format the authors show that clade-D ARFs act as regulators of physiologically significant auxin signaling in moss and also present a plausible model for their mode of action. Taken together, this is a major contribution of considerable news value with potential to attract the interest of colleagues studying plant development, land plant evolution and auxin function.The authors should consider the following comments and suggestions to further strengthen the manuscript (ordered according to structure of manuscript rather than priority):1. I have no questions, comments or suggestions relating to the title, abstract, or introduction.2. Relating to Fig 1:a. The authors could consider to remove narC18 and narD157 from the figure since the more severe phenotypes of these lines are not primarily related to mutations in ARFd genes.b. The phenotypic deviation in presence of NAA in narD122, nard122/Δarfd1, and Gd/NLS4Δarfd1 (marked by arrow heads) cannot really be seen. The authors should include inserts of gametophores at higher magnification.c. Why does the DR5:DsRed2 output appear much stronger in Gd/NLS4Δarfd1 than narD122 and nard122/Δarfd1? Please comment.d. I suggest that also DR5:DsRed2 output in the absence of NAA is shown. Please add or explain why it is not needed.3. Relating to Fig S1:a. The results section states that 'we used CRISPR/Cas9 to delete ARFd2 in the WT and Δarfd1 background', but when I look at Fig S1A I get the impression that ARFd1 was targeted in a pre-existing Δarfd2-8 background. Please change or clarify.b. I would have liked to see the phenotypic characterization presented in Fig S1B-D and Fig 2 repeated for more independent single and double mutant lines. If such data exist, please include it. If not, I still find it acceptable in this particular case since additional and partly supporting phenotyping data is presented in other figures for other mutations in the same genes.4. Relating to Fig 3:a. In the results section discussing Fig 3b it is stated that 'arfd1T653L-mYpet lines exhibit an auxin sensitivity that is intermediate between the control and Δarfd1,d2 lines'. This is not very obvious from the data. Please provide statistical support for this claim.b. Fig 3C indicates lower gametophore counts in arfd1Y543*-mYPet Δarfd2 lines than in the Δarfd1,d2 lines. Is there an explanation for this?5. Relating to Fig 4:a. The finding that ARFds are required for caulonemal development only in nutrient-rich conditions is interesting. Can it imply that ARFd serves as a modifier of auxin responsiveness in response to nutrient availability? Maybe it could be worth to check if the expression of ARFd (and possibly auxin regulated genes likely to act downstream of ARFd) is regulated by the nitrogen source?6. Relating to Fig 5 and the corresponding results section:a. The data supporting that auxin affects the timing of branch initiation in Physcomitrium appears very solid. Still, after a quick look at Johri and Desai (1973) my impression is that their claim is that the proportion of caulonema-like branches is increased in response to auxin in Funaria, rather than the number of branches or the timing of branching. I may be wrong, but the author may want to comment on this.b. The data presented appears to clearly support that branch initiation is delayed in the Δarfd1,d2 lines both in absence and presence of external auxin, but it is not as obvious if the ARFd genes are important for mediating the auxin-induced change in timing of branch initiation. The authors may want to provide statistical support for such a claim.7. Relating to Fig 6:a. The strong ARFd expression in protonemal cells at the base of gametophore initials is intriguing. An obvious question that comes to mind is if ARFd expression maxima in certain protonemal cells marks where branches differentiating into buds/gametophores will emerge. It is not a trivial experiment, but if the authors could provide data supporting such a function by some kind of time-lapse study it would be a major contribution.8. Relating to Figure 7:a. In the results it is stated that ARFd1 interacts with both ARFa4 and ARFa8, but I cannot find data supporting the former claim.b. In the results it is stated that ARFd1ΔDIV retains the ability to interact with ARFa8 but not with ARFb1, but I cannot find data supporting the latter claim.9. I find the discussion clear, relevant and to-the-point. I don't have any questions, comments or suggestions relating to the Material and Methods section.Rev. 3: Dolf Weijers – note that this reviewer has signed his reviewBascom Jr and colleagues describe a genetic analysis of the Physcomitrium patens D-class ARFs. This is a mysterious subclade of the ARF transcription factor family  that exist in bryophytes and lycophytes, but were lost in ferns and seed plants. A prior study in Marchantia showed that a D-class ARF mutant has reduced auxin response, but no detailed analysis was perfoemd, and no mechanism has been established. The authors of the present manuscript identified a D-class ARF mutant in a screen for auxin-resistant Physcomitrium mutants, and explore its role in auxin response. Overall, this reports important findings that are of importance for understanding the principles underlying auxin response. However, there are a number of points that require further exploration, clarification or elaboration.1. The authors present a role of ARFds during P. patens development. However, unlike the authors suggest in the abstract, it does not seem to be "major role", given that this occurs only under specific nutritional conditions.2. While the "ARFd" nomenclature was coined by the authors , the term D-class has not been widely used. Instead, ncARF  has been used in the literature. I encourage the authors to explicitly state that this refers to the same proteins and propose the future used of only one name (ARFd).3. The interest in D-class mutations is clear, but the reader can be left wondering about the other mutations found in the mutagenized lines, as those in A and B-class ARFs, PINc or RSL7. I suggest to either leave out the information on the (putative) identity of these other mutants, or provide information on the molecular lesions, especially given the difference in the visible phenotypes in Fig.1 for narC18 and narD157.4. Similarly, while the authors perform CRISPR against ARFd1 in the narD122 line, this line alone is not proven to be complemented with a ARFd1 WT copy, so other mutations, that most likely have occurred due to the technique used, may be important for auxin sensitivity (maybe epistatic to ARFd1?). What other mutations were found in narD122?5. The authors make single and double arfd mutants, but quickly jump to chlorophyll content analyses. Are these mutants similar or different to WT? how do they look in BCD and BCDAT with or without different concentrations of auxins? Some of this data is in supplemental material but is never described or discussed.6. Image quality in Fig. 1 and 2 is poor. Please provide better pictures, maybe higher magnification would convey the phenotypes better. This would help the reader in following authors´ interpretation for auxin resistance.7. Authors only show DR5:dsRED expression in response to NAA. In order to properly compare DR5:dsRED expression it would be important to see DR5:dsRED activity on medium without auxin, and ideally also with IAA (rather than NAA).8. In some cases, the DR5 line shows a somewhat different behaviour in auxin response compared to a WT. For example, the DR5 strain shows a much smaller reduction in chlorophyll content (22% vs 36%) compared to the WT strain. The authors describe this as "similar", but this difference is striking and not trivial. Also, together with other findings, it seems that this background has inherent auxin insensitivity. Is this something common in Reute/Grandsen cross progenies? Why not making mutants in a non-DR5 background since the responsiveness seems to be dampened here?9. In conjunction with the comment above, I have some issues with using chlorophyll content as a way of quantifying auxin response. The authors mention later that arfd1,2 mutations lead to delays in gametophore formation and protonema differentiation on BCDAT. Doesn't this fact introduce a bias in the chlorophyll analysis? Ater 2 weeks, the composition of tissues in the mutants is very different form wild-type . These different tissues would contribute to the overall chlorophyll content per gram of tissue in a unique way. Are these stages comparable?10. It is unclear why the authors did not include the original arfd/nard or arfd2 single mutants in the gene expression analyses . These single mutants are disregarded also from Fig. 3 on. Also, why using such a long IAA treatment (4.5 hours) to measure immediate auxin responsive gene activation? The authors indicate that ARFd1 has a larger contribution to the auxin response than ARFd2. But this is entirely bound to the first chlorophyll content analysis and single mutants are not checked for any other analysis. Is there any specific reason?11. The results suggest that ARFd proteins are involved in altering growth patterns in response to changing nutrient conditions. To provide mechanistic understanding, it would be important to know if (and how) ARFd expression and protein levels are modulated by the nutrient contrasts used here.12. ARFd1-mYPet expression seems to be very similar between chloronema and caulonema tissues. Since ARFd1 appears to also have an impact on timing of branch formation, it would be interesting to know if ARFd1 expression levels are different in the apical and subapical cells, and how this correlates with branch formation. Authors also claim that YPet is not detected in mature gametophores, but the picture is not clearly showing that, especially at this magnification, since it could still be expression at the basal parts of the mature gametophore.13. An open question is the molecular mechanism by which ARFd acts. One option is that it supports transcriptional activation by A-class ARFs, another is that is acts as an Aux/IAA scavenger . The authors clearly favor the former throughout the text, and do not comment on the possibility of the latter at all. I would encourage the authors to use e.g. transient gene expression assays in protoplasts to test these two hypotheses. Otherwise, the existence of a second hypothesis should be mentioned in the discussion, and the claim of co-activation activity toned down.14. In page 20, the authors write: "This is the first evidence in any system that ARF oligomerization is important for activity.". This statement is not entirely correct. Kato et al 2020 showed that the MpARF1 PB1 domain is essential for function, and that it can be replaced by a heterologous oligomerization domain. I would suggest removing this statement.15. At the end of page 21, the authors write: "…most models suggest that ARF-ARF interaction occurs primarily via the dimerization domain adjacent to the DBD, and only when the proteins are bound to DNA ". This is also not entirely correct, ARFs dimerize through their DBD also in the absence of DNA. Moreover, a recent preprint showed that the DBD and PB1 act cooperatively in promoting dimeric DNA binding, indicating that both domains contribute to dimer strength.16. The text needs some refinement to improve clarity:o In the introduction, the authors refer to the DNA binding sequence of ARFs. While the shown sequence is indeed an ARF binding motif, this not the only one. This may lead to confusion for a general audience. I suggest to reword to auxin responsive elements, but given these specific piece of data adds nothing to the rest of the work, the sentence could be deleted.o Authors introduce only A- and B-class at the beginning but then refer to C-class. This can also be wrongly interpreted.o Mutant nomenclature is not clear. While the single alleles are named properly in Fig. S1 (as with Δarfd2-8), the double mutants obtained are also dashed in the text and figures . This dash is misleading, especially since it refers to the ARFd1 allele.o Authors indicate in page 12 the use of NAA for the screening, but later in page 13 explain why they use NAA instead of IAA. This should be explained at the very beginning.o On page 14 the authors mention Figure 3C, D but Fig. 3D does not exist .o On page 13 the authors say that "Previous reports that auxin treatment results in down regulation... photosynthesis", please provide a reference.o On page 14 the sentence "To gain insight...narD122" would read easier if the authors used the term "mutation" instead of "mutant".o In Figure 4 it would be much easier to interpret results if the type of medium used to grow plants is mentioned IN the figure (BCD resp. BCDAT) instead of in the figure description.o Figure 5 D, G and J contain the same data replicated. While I guess this done for clarity, please explain or indicate this in the captions for the sake of transparency.o Unmerge K-S tests in Fig. 5G and 5H for each of the mutant lines. Each line seems to behave differently (in mock conditions).o In general, captions are quite uninformative and lack data for proper figure interpretation such as age of plants, time of treatments, etc. Statistical tests, number of samples, and replicates should be shown as well. 4 Apr 2023AttachmentAuthorResponse_ARFd_v3.docxSubmitted filename: Click here for additional data file. 25 Apr 2023Dear Mark,Thank you for your patience while we considered your revised manuscript entitled "Clade-D Auxin Response Factors Regulate Auxin Signaling and Development in Physcomitrium patens" for publication as a Research Article at PLOS Biology. 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Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are essential for readers to assess your analysis and to reproduce it:Fig. 2B-E; Fig. 3B, C; Fig. 4B-E; Fig. 5H; Fig. 6B; Fig. 8B, C; Fig. S2B-E; Fig. S3D; Fig. S4B-E and Fig. S5CNOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived .Please also ensure that figure legends in your manuscript include information on WHERE THE UNDERLYING DATA CAN BE FOUND, and ensure your supplemental data file/s has a legend.Please ensure that your Data Statement in the submission system accurately describes where your data can be found.------------------------------------------------------------------------Reviewers' commentsRev. 2:All my requests and suggestions on the original submission have been addressed in an acceptable way.Rev. 3: Dolf WeijersThe authors have done an excellent job in responding to my comments and revising the manuscript. I look forward to seeing this paper in print. 4 May 2023AttachmentAuthorResponse_ARFd_v3.docxSubmitted filename: Click here for additional data file. 12 May 2023Dear Mark,Thank you for the submission of your revised Research Article entitled "Clade-D Auxin Response Factors Regulate Auxin Signaling and Development in the moss Physcomitrium patens" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Xinnian Dong, I am delighted to say that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. 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Propolis has high potential beneficial bioactive properties such as anti-oxidative, antimicrobial, and anti-tumour activities. However, the bitter taste and the insolubility nature of propolis in water lead to some limitations in their usage in functional food applications.Herein, we evaluated the effects of nanoencapsulation of propolis at the different concentration levels  into the polyvinyl alcohol (PVA) nanoparticles using the electrospraying method, on the structural, physical, antioxidant, antimicrobial and thermal properties.S. aureus at low concentration ratios, whereas it had no inhibitory activity against E. coli O157:H7.The results revealed that the fabricated nanocapsules (PVA-NPs) obtained under optimal conditions had uniform size distribution and unstable particles with small particle size between 104-258 nm, a polydispersity index <0.317, and a zeta potential between -5 and +5 mV. The maximum encapsulation efficiency of PVA-NPs was about 25.32 % for 1 % of the initial propolis loading level. DSC thermal experiments showed an increase in the thermal stability of the propolis loaded PVA nanoparticles as compared to the neat PVA nanoparticles. The percent inhibition of DPPH radical scavenging activity of the nanocapsules was between 80 and 89 %. SEM analysis revealed that PVA-NPs had a spherical shape with a rough surface and were composed of long and thin fibres at nanometric diameters. FT-IR analysis showed that no indications of any chemical reactions were found between the constituents of the core and wall material due to their physical mixing. Antibacterial efficacy was evaluated by the Broth dilution method and PVA-NPs exhibited good inhibitory activity against PVA-NPs fabricated using the electrospraying technique can be used for the development of a new promising natural and bioactive agent in the food and pharmaceutical industry. Therefore, nanotechnology has become one of the most efficient and popular topics with a wide range of application fields such as medical, agricultural and food science. One of the most common research and development areas in nanotechnology applications in the food and pharmaceutical industry is the encapsulation of bioactive compounds. Encapsulation can be defined as a process that is the entrapment or coating of a pure material or a mixture  into a coating material  ,2. The eundings) . This teundings) .Polyvinyl alcohol (PVA) is a water-soluble, odourless, tasteless, non-toxic, non-carcinogenic and fully biodegradable synthetic polymer obtained by partial or complete hydrolysis of polyvinyl acetate. It forms an excellent particle and fibre structure either alone or when blended with other natural polymers and offers a great promise for better biocompatibility, particularly due to its good chemical resistance, stability and flexibility. Also, it shows stability in a wide range of temperature and pH ,7.Propolis (bee glue) is a natural honeybee product with a naturally heterogeneous complex matrix used for the construction, maintenance, insulation, and protection of hives by the honeybees from various plant sources . It is aRaw propolis used in this study was obtained from Arifoğlu Spice& Food Industry Trade Company . Polyvinyl alcohol , nutrient broth , Maximum Recovery Diluent  and 2,2-Diphenyl-1-picrylhydracyl (DPPH)  were purchased. Methanol and ethanol were analytical grades.Propolis extract was obtained by using 80 % ethanol as solvent. Firstly, 8 % of ethanolic propolis solution was prepared. The obtained solution was stirred with a magnetic stirrer  for 24 h at room temperature and then kept at -20 °C for 3 days to precipitate the impurities. After that, the supernatant was taken and filtered through filter paper. Following the filtration, the solution was first centrifuged  twice at -5 °C for 5 min at 4.200 rpm. Afterwards, the supernatant was filtered by passing through a 0.22-μm filter. Removal of the solvent from ethanol extract was done by vacuum evaporation  at 50°C. The resulting propolis extract was kept in the refrigerator at 4 °C until further use.In this study, prior to propolis encapsulation, optimum PVA concentration was determined to obtain homogeneous and low-diameter PVA nanoparticles using the electrospraying technique. For this purpose, different PVA solutions were prepared at various concentrations to determine the most appropriate PVA concentration and to optimize the electrospraying conditions and then the nanoparticles were fabricated depending on the different processing conditions using the electrospraying method. In light of the obtained results of the size and zeta potential measurements, optimization studies were performed.-1), and the tip-to-collector plate distance (10-20 cm), were tested. The most suitable processing parameters were determined by measuring the particle size and assessing the microscope images of resulting nanoparticles applied to the different process parameters mentioned above.Different aqueous PVA solutions 5 to 10 % (w:v) were prepared and each solution was shaken in a water bath  at 100 rpm for 20 min at 50 °C to form a homogeneous solution. The nanoparticles were fabricated using an electrospraying device . For this purpose, 5 mL plastic syringes , each filled with neat PVA feed solutions, were inserted into the syringe pump  to spray the feed solutions. During electrospraying, different process parameters, such as voltage (10-20 kV), solution flow rate  was used to determine the variations in the particle size and polydispersity index (PDI) values depending on the PVA concentration in the feed solutions . For this purpose, the nanoparticles were diluted with sterile distilled water, homogenized using an Ultra-Turrax  for 30 min, and then passed through 0.45 μm pore size disposable filter. Measurements were carried out in triplicate.et al. (2007) [A slightly modified procedure reported by Durán . (2007)  was follζ-potential were presented as nanometre and millivolt, respectively.Particle size and zeta potential measurements of PVA-NPs were measured using a Malvern Zetasizer Nano ZS analyser  at 25 °C. For this purpose, PVA-NPs were diluted with distilled water and the measurements were conducted. Each result was calculated as the average of 10 (one set of 10) measurements. The results of particle size and et al. [The encapsulation efficiency of PVA-NPs was determined according to a method described by Durán et al. , with so-1, resolution: 4 cm-1, and scans: 32).Molecular characterization of the propolis extract and PVA-NPs were performed using a Bruker FT-IR spectrometer model Tensor 27  coupled to a diamond platinum ATR unit  in the temperature range of 40-400 °C at a heating rate of 10 °C minFirstly, 1.5 mL of ethanolic extract of PVA-NPs was well-mixed with 1.5 mL of DPPH solution, vigorously vortexed for 1 min and left in a dark environment for 30 min at room temperature. Then the absorbance values were measured at 517 nm using a UV-visible spectrometer . The DPPH scavenging activity was calculated using the following equation:et al. [S. aureus (ATCC 25923) and Escherichia coli O157:H7 (ATCC 33150)). Initially, after activating bacterial strains twice in nutrient broth at 37 °C for 24 h, the test tubes, including 7 mL of nutrient broth, were seeded with the bacterial strains  and then incorporated with2 % of the PVA-NPs samples. Following incubation of the test tubes for 5 h in the shaking incubator at 37 °C at 140 rpm, serial dilutions were prepared, and colony counts were analysed using a spread plating method. Colony forming units were counted after 24 h of incubation of Petri plates at 37 °C. The results were presented as logarithmic values.The Broth dilution method described by Feyzioglu et al.  was usedThe surface morphology of PVA-NPs was analysed by scanning electron microscopy  under vacuum at 20 kV and at a 7.6 mm of working distance. Prior to analysis, the samples were lyophilized and coated with gold.t-test at the p<0.05 level and the statistical analysis of data was performed by the JMP software .A minimum of three independent experiments with two replicates were performed to achieve efficient results and the findings were tabulated as mean ± standard deviation. A one-way ANOVA  test for the determination of the statistical differences between the data was applied using a In this study, firstly, different concentrations  of PVA solutions were fed to the electrospraying system. The particle size of the nanoparticles was monitored. The highest particle size value was measured in the solution containing 10 % PVA (212-255 nm), followed by the solution containing 5 % PVA (144-180 nm), and the solution including 6 % PVA (86-106 nm). Microscope images showed the formation of nanofibers at higher concentrations than 7 %, while dark (black) beads were obtained at lower concentrations (5 %). Moreover, laboratory trials showed that an increase in the ultrasonication time before the electrospraying led to the formation of more homogeneous particles. Overall, ultrasonication frequency and coating solution concentration were determined as 50 Hz and 6 %, respectively, in this study. Moreover, optimum electrospraying conditions were an applied voltage of 15 kV, a tip-to-collector plate distance of 10 cm and a solution flow rate of 0.5 ml / h.Following the optimization of electrospraying conditions, PVA-NPs were prepared with the incorporation of propolis extract. Then the resulting nanoparticles were characterized.e.g., particle size, encapsulation efficiency, oxidative stability, and morphology) [The summary of the ζ-potential, PDI and mean particle size findings for the PVA-NP0, PVA-NP1, PVA-NP2, PVA-NP3 and PVA-NP were given in phology) .PVA-NP0 had lower average particle size values (106.37 nm) compared to the other formulations. The average particle size of PVA-NPs loaded with the aforementioned five concentrations of propolis was found to be between 106.37 and 258.51 nm. Moreover, an increase in the loaded propolis ratio led to an increase in the average particle size except for PVA-NP4. However, no significant differences were found between PVA-NP1, PVA-NP2 and PVA-NP4 (p>0.05).ζ-potential values were in the -0.11 to 3.09 mV range with no statistically significant influence of propolis ratio between PVA-NP0/PVA-NP1 or PVA-NP2/PVA-NP3/PVA-NP4 (p>0.05). In the current study, PVA-NPs had zeta potential values near zero with slight differences depending on the propolis concentration. Increasing propolis caused a more negative surface potential, while the highest ζ-potential was measured in PVA-NP0 (3.09 mV). The ζ-potential values between -30 and +30 mV are considered as the formation of stable water suspensions [ζ-potential was lower (more negative) as the propolis ratio increased, but the variations were not very high. Similar findings were reported by Baysan et al. [ζ-potential values of the propolis-loaded capsules prepared with different coating material combinations  in the range of -6.16 to 0.42 mV. The reduction in diameter from PVA-NP4 to PVA-NP0 was probably due to the changing surface charge of the PVA-NPs and the agglomeration of nanoparticles in an aqueous medium.The ζ-potential is a very good index for the specification of the surface electrical status (the electrostatic or charge repulsion/attraction) of colloidal systems. This parameter was used for the evaluation of the encapsulation of the core material in the nanoparticle and the stability or aggregation of the nanoparticle in the suspension. All the pensions , showingn et al. , who foue.g., stability, the release of core material and encapsulation efficiency) [et al. [p<0.05), but PVA-NP3 and PVA-NP4 exhibited statistically no significant differences (p>0.05). Additionally, PVA-NP0 and PVA-NP2 had a more homogenous particle size distribution as compared to the other PVA-NPs. In a previous study, particle size (200-280 nm) on a nanometric scale, the ζ-potential values (-12.7 to -33.5 mV) and PDI (0.089 to 0.169) for the polymeric nanoparticles loaded with Brazilian red propolis extract were determined by Do Nascimento et al. [ζ-potential (-26.9 to -31.7 mV) of propolis-loaded zein/caseinate/alginate nanoparticles [PDI is one of the best indicators to evaluate the particle size distribution in suspensions and estimate the physical and rheological features of the colloidal systems (iciency) . Lower P [et al. . The useo et al. . One recarticles . Our resarticles .p<0.05) and the lowest (5.09 %) encapsulation efficiency values, respectively  compared to the reference core amount incorporated into the encapsulation system. PVA-NP3 and PVA-NP1 had the highest (25.32 %)  [-1. The propolis extract spectra also exhibited the band’s disappearance at 1043 cm-1 from PVA-NPs. Furthermore, corresponding to the PVA-NPs, no new bands were determined. In the FTIR spectra of the propolis extract, the wavelength around 1045 cm-1 shifted to the higher band (1083 cm-1) with the fabrication of PVA-NPs. Moreover, molecules containing the plane of aromatic C-H regarding the angular deformation were attributed to near 877 cm-1 [et al. [The wavelengths found at 1725-1705 cm ketones . The banic rings . The obsbending) . Additio877 cm-1 . Similar [et al. . OverallTg), onset temperature (To), peak temperature (Tp) and ending temperature (Te) values were determined. As can be seen, while the DSC thermographs of PVA-NP1, PVA-NP2, PVA-NP3, and PVA-NP4 were similar, whereas PVA-NP0 possessed a broader endothermic peak from 242 °C to 390 °C with a maximum temperature of 305 °C. It can be due to the changes in thermal characteristics of PVA with the addition of propolis. The PVA-NP0 depicted the three endotherm events with To at 44, 150 and 242 °C, Tp at 105, 225 and 305 °C and Te at 150, 237.5 and 390 °C and its corresponding Tg was determined at 50 °C.The thermographs of PVA-NP0, PVA-NP1, PVA-NP2, PVA-NP3, and PVA-NP4 were illustrated in et al. [To of the third endothermic peak for PVA-NP0 shifted to higher temperatures after encapsulation of propolis, which implied that encapsulation supplied thermal protection of propolis. Variations of peak positions indicated the interactions between PVA and propolis extract that led to the formation of new structural organizations. Among the tested PVA-NPs, in general, PVA-NP1 and PVA-NP4 exhibited similar endothermic peaks with lower temperature values, while PVA-NP3 showed endothermic peaks at greater degrees . Moreover, the DSC thermogram of the PVA-NP2 illustrated four endothermic peaks at Tp of 40, 128, 223, and 329 °C. Nascimento et al. [Encapsulation of propolis extract into the PVA polymer matrix resulted in four endothermic peaks. Our findings supported the results obtained by do Nascimento et al. , who alsp>0.05). Our findings were supported by the results obtained by Aytekin et al. [The DPPH scavenging activity  of the Pn et al. , who repn et al. , DPPH inE. coli O157:H7 and S. aureus) and the findings were given in S. aureus, where a complete elimination was achieved by the effect of PVA-NP2, PVA-NP3 and PVA-NP4. A significant reduction in E. coli numbers was also provided by the application of propolis-loaded PVA-NPs. In a previous study, Przybyłek and Karpiński [The antibacterial activity of PVA-NP0, PVA-NP1, PVA-NP2, PVA-NP3 and PVA-NP4 were tested against two pathogenic bacterial strains  that may directly influence the cell wall of the bacteria [The results also demonstrated that propolis extract kept its antibacterial activity after nanoencapsulation. The findings regarding earchers ,38. Simiearchers  reported [et al. , who repbacteria .SEM images of the PVA-NPs are shown in Additionally, another possible reason could be due to the high viscosity of the coating material. Earlier studies reported that the increase in the viscosity of the solution resulted in spindle-like droplets with pure fibres . SEM exaS. aureus than E. coli. SEM images demonstrated that the nanoparticles had a spherical shape with a rough surface and were composed of long and thin fibres with nanometre thickness. Overall, the current work confirmed that nanoencapsulation of propolis using electrospraying technique could offer a new and effective approach to overcome its limitations for utilization in the food and pharmaceutical industries.Propolis has been popular in recent years for its health-related and pharmaceutical properties. However, it cannot be consumed in its original form due to its bitter and unpleasant taste. Its insolubility nature in water and the easy loss of its antioxidant activity and phenolic compounds are other limitations of propolis in the food industry. In the current work, propolis at different concentrations  were encapsulated into PVA polymer matrix using the electrospraying method. The dynamic light scattering analysis showed that the nanoparticles had nanoscale particle size with moderate PDI values and formed unstable colloidal dispersions. Encapsulation efficiency of up to 25 % and high DPPH scavenging activity of up to 89.25 % were found for propolis-loaded PVA nanoparticles. FT-IR analysis showed the successful propolis encapsulation and interaction between the constituents. DSC analysis revealed that the thermal stability of propolis-loaded PVA nanoparticles was higher in comparison to their neat nanoparticle forms. Regarding antibacterial activity, the loaded nanoparticles showed stronger antibacterial activity against


Q lasers. However, it is still difficult to realize from conventional conjugated polymers. Herein, we create the molecular super-hindrance-etching technology, based on the π-functional nanopolymer PG-Cz, to modulate multilayer cracks applied in organic single-component random lasers. Massive interface cracks are formed by promoting interchain disentanglement with the super-steric hindrance effect of π-interrupted main chains, and multilayer morphologies with photonic-crystal-like ordering are also generated simultaneously during the drop-casting method. Meanwhile, the enhancement of quantum yields on micrometer-thick films (Φ = 40% to 50%) ensures high-efficient and ultrastable deep-blue emission. Furthermore, a deep-blue random lasing is achieved with narrow linewidths ~0.08 nm and high-quality factors Q ≈ 5,500 to 6,200. These findings will offer promising pathways of organic π-nanopolymers for the simplification of solution processes applied in lasing devices and wearable photonics.In situ self-assembly of semiconducting emitters into multilayer cracks is a significant solution-processing method to fabricate organic high- In particular, organic high-Q random lasers are a crucial step toward electrically pumped lasing grid}-co-{grid} (PODPFG) [nglement ,37, whicate Fig.  also conate Fig. . In contate Fig. , which aWe further simulated the self-assembly behaviors of PG-Cz to predict the feasibility of forming multilayer structures, based on the established aggregate structure as the deposited substrate. Such deposited aggregate displays a relatively rough surface with vacancy sizes of 1 to 3 nm, due to the presence of bulky repeat units and torsional main-chain conformation . In Fig. θ from 0° to 60°. Via simulating the interference fringe . The above results suggest the presence of air layers (n ≈ 1) in the crack regions. Notably, these interface cracks can serve as a natural refractive-index inhomogeneity (the refraction index contrast ∆n ≥ 0.8) to facilitate photonic reflection and lasing action along the in-plane direction of the film surface.We further demonstrated the multilayer crack morphologies through angle-resolution reflectance spectra  to induce a strong Purcell effect, the factor of microcavity effect  from mulI0–0/I0–1) can be relatively increased when elevating the temperature  on thick multilayer films  is slightly lower than that of solvent-casting (I0–0/I0–1 = 0.26 to 0.31) and solvent-annealing films (I0–0/I0–1 = 0.28 to 0.33) in the temperature range of 80 to 300 K, revealing the higher order of H-aggregate emission from the drop-casting method.Furthermore, temperature-dependent photoluminescence spectra (Tem-PL) are used to investigate the aggregate emission feature of PG-Cz in multilayer crack morphologies. In Fig. ure Fig. F. The abure Fig. , verify lms Fig. . Thus, t−2, and the threshold is achieved as 23.5 μJ cm−2  can also be maintained, which exceeds general random lasers  of conjugated polymers [We examined the application of multilayer crack morphologies in lasing actions, through 343-nm pulse pumping in the air. A pulse length of 1 ns, which is 3 to 4 times longer than the average fluorescence lifetimes Fig. , was selm−2 Fig. . The fulpolymers ,19.Q lasing actions and multilayer crack morphologies. Through power Fourier transform (PFT) of lasing spectra  of 50 to 300 μm according to the equation Lc = 2πd/n. These cavity sizes are roughly within or consistent with the length of discrete multilayer films between two crack interfaces, reflecting the in-plane direction of lasing oscillation (perpendicular to the multilayer cracks). Furthermore, we performed the finite-different time domain (FDTD) simulation to confirm the role of crack regions in optical behaviors. In Fig. −2, and a gain coefficient of 25 cm−1  was prepared as the controlled morphology, which also exhibits massive material/air interfaces like cracks but does not have mesoscale-ordered multilayer architectures  was prepared, and then the drop-casting (dropping solution onto the quartz substrate and then evaporating toluene solvent in 5 min), solvent-casting (transferring into plant vessel and slowing evaporating toluene solvent in 4 days), and solvent-annealing methods (depositing the PG-Cz-based microsphere film onto the quartz substrate under filling toluene atmosphere in 1 to 2 days) were used for the self-assembly into multilayer crack films, similar to our previous work [PG-Cz and PODPFG were prepared according to our previous work ,38. PG-Cous work .DP = 10 on each single-chain) are constructed in an amorphous cell to perform molecular dynamics simulation under a number–pressure–temperature constant (NPT) ensemble (fixed with 298 K temperature and 10−4 GPa pressure) and COMPASS force field . Then, we perform the NPT dynamics simulation of PG-Cz aggregates under 298 K temperature, 1.5 fs time step, and 1,500 ps total time, in which each conformation information  is extracted at the interval of 7.5 ps. For the simulation of PG-Cz chains with toluene or ethanol solvent molecules, we establish corresponding amorphous cells with merely PG-Cz single chains , merely toluene or ethanol molecules (with 40 molecules), and the mixing systems with PG-Cz chains  and solvent molecules , each of which was set at a density of 0.4 g cm−3. All of the above systems undergo geometric optimization under the same conditions as those of PG-Cz aggregates . Then, NVT dynamics simulation was performed under the COMPASS force field . For the molecular dynamics simulation of self-assembly at the interface (NVT ensemble). The time step is set as 1.5 fs, and the total time is set as 3 ns, where each conformational information is extracted at the interval of 12 ps. For the simulation of stretching polygrid entanglement aggregate (using Forcite models), the input stress is set as 0, 2, 4, 6, 8, 10, and 12 GPa, respectively, along with the NPT ensemble, Souza-Martins Barostat, and Nose-Hoover-Langevin thermostat. The calculation of microscopic stress on each plane direction  is performed under the NVT ensemble.All molecular dynamics simulations are performed through the Forcite modules in the Material Studio 2016. For the simulation of aggregation models, 4 PG-Cz chains (with λ = 1.54050 Å). The highly ordered multilayer microstructures of PG-Cz-based solvent-casting and solvent-annealing films were also examined by the POM characterizations with NIKON LV100ND systems.The GPC spectral of PG-Cz oligomers and long single chains were obtained through an HP1100 HPLC system equipped with 7911GP-502 and GPC columns (polystyrene as the standard and tetrahydrofuran as the eluent). The detection of conformational states in self-assembled solid is based on the Raman spectra with a Horiba evolution spectrometer, which is coupled to an Olympus BH-2 confocal microscope (the excitation source is based on the He–Ne laser with the 633-nm line). The microspheres of PG-Cz-based microsphere films were observed by an inverted fluorescence microscope 278 , as a demonstration of its blue-emitting feature. The large-scale morphologies are observed by profile (Bruker) and SEM . TEM  and HRTEM  were also applied to further testify the self-assembled morphologies and selected-area electron diffraction. X-ray diffraction was performed on a Bruker D8 x-ray diffractometer with Cu KR radiation (θ = 0° to 60°) is identical to the detected reflectance angle. The aluminum mirror serves as the standard. The elimination of the background scattering is based on the black sponge, which transforms the count mode spectra into the reflectance spectra with absolute reflectance values. In addition, the transmittance (To) of various PG-Cz-based films is obtained through the transformation from the absolute absorbance (Ao), according to the equation Ao = −lg To. The absorbance can be obtained via the ultraviolet spectra from LAMBDA 35 systems. The simulation of optical interference fringe is performed by MATLAB software .The light–matter interaction of these multilayer crack morphologies was characterized by the angle-resolved spectrum system with micro-region . The visible light source (with emissive wavelength centered at 550 to 750 nm) is the halogen lamp. We used the constant angle of incidence and reflection mode as the test mode, during which the incidence angle . The integral sphere was used to collect the photons including the reflected types from the xenon arc lamp and the emissive types from PG-Cz chains. The Φ values can be evaluated by the ratio of emissive  to absorbed (Ia) numbers of photons from PG-Cz chains, where the Ia values can be obtained by the differences of reflected photons between the quartz background  and the film samples . Thus, the Φ values can be obtained via the equation Φ = Ie/(Ib − Is). To improve the testing accuracy of collected photons, all processes of the photonic collection were performed by repeating 2 cycles. The fluorescence lifetime of various PG-Cz-based films was detected by the spectrometer system (Princeton Instrument Acton SP2500I) that is equipped with a streak camera (Optronis Optoscope sc-10), based on the excitation through a 325-nm femtosecond pulse laser. Tem-PL was employed by the 355-nm diode laser  to afford the photoluminescence spectra under the environment temperature varying from 80 to 300 K, where the liquid nitrogen cryostat was used to maintain the environment at ultralow temperature (80 K).The deep-blue emission and 2-dimensional PL spectra of various PG-Cz-based films were detected through RF-6000 Plus systems. The The pump source  with an excitation wavelength of 343 nm, a pulse width of 1 ns, and a repetition rate of 100 Hz were selected for detecting random lasing. The spectra of ASE and lasing are detected by an optical spectrometer  that exhibits a resolution of ~0.03 nm. The gain coefficient of ASE and lasing is based on the variable stripe length measurement.X–Z coordination. The multilayer crack film is set as the model with a length of 100 μm (covering the X coordination of −50 to 50 μm) and a total height of 3 μm . The refractive index of the film region is set as n = 2.1 according to the angle-resolution reflectance spectra. On 2 sides of interface cracks, the virtual region  is added with a tunable refractive index  to investigate the influence of refractive index contrast of interface cracks on-field intensity at the ends. The model of microsphere film is established by the same total sizes with 300 microspheres . The light source is selected with the wavelength centered at 439.3 nm (consisting of the dominant lasing or ASE or luminescence wavelength). The detection of light leakage intensity is based on the 2-dimensional monitor established above the film surface initially at 0.2 μm.The FDTD simulation is performed, where the simulation is based on the